No abstract

We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.

Efficient polyketide synthesis derived from plasmid-borne heterologous Streptomyces polyketide synthase (PKS) gene clusters necessitates a suitable host strain. Well-characterized laboratory strains such as Streptomyces coelicolor or Streptomyces lividans and their frequently used derivatives carry endogenous genes for the synthesis of actinorhodin (among other PKS genes), which might interfere with the efficient production of extrachromosomally encoded PKS proteins and the quantitative analysis of their secreted polyketide products. To circumvent this problem, a frequently used S. coelicolor derivative, designated CH999, was engineered to lack most of the actinorhodin gene cluster. However, this strain can only be transformed with methyl-free DNA. Additionally, unlike its otherwise isogenic parent CH1, CH999 exhibits low transformation efficiencies. Here, we report the construction of two S. lividans host strains, K4-114 and K4-155. With respect to the actinorhodin gene cluster, both are genotypically identical to CH999; however, both can be transformed at considerably higher frequencies and also with methylated DNA. Upon transformation with the appropriate expression vector, CH999, K4-114 and K4-155 all produce the erythromycin precursor 6-deoxyerythronolide B (6-dEB) equally well.

The extent to which human immunodeficiency virus (HIV) type 1 drug resistance compromises therapeutic efficacy is intimately tied to drug potency and exposure. Most HIV-1 protease inhibitors maintain in vivo trough levels above their human serum protein binding-corrected IC(95) values for wild-type HIV-1. However, these troughs are well below corrected IC(95) values for protease inhibitor-resistant viruses from patients experiencing virologic failure of indinavir and/or nelfinavir. This suggests that none of the single protease inhibitors would be effective after many cases of protease inhibitor failure. However, saquinavir, amprenavir, and indinavir blood levels are increased substantially when each is coadministered with ritonavir, with 12-h troughs exceeding corrected wild-type IC(95) by 2-, 7-, and 28-79-fold, respectively. These indinavir and amprenavir troughs exceed IC(95) for most protease inhibitor-resistant viruses tested. This suggests that twice-daily indinavir-ritonavir and, to a lesser extent, amprenavir-ritonavir may be effective for many patients with viruses resistant to protease inhibitors.

Amprenavir (Agenerase, 141-W94, VX-478) is a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PRI) recently approved for the treatment of HIV-1 infection in the United States. A major cause of treatment failure is the development of resistance to PRIs. One potential use for amprenavir is as salvage therapy for patients for whom treatment that includes one (or more) of the other four currently approved PRIs-saquinavir, indinavir, ritonavir, and nelfinavir-has failed. We evaluated the cross-resistance to amprenavir of viruses that evolved during treatment with the two most commonly prescribed PRIs, nelfinavir and indinavir. Unexpectedly, a dramatic increase in susceptibility (2.5-to 12.5-fold) was observed with 20 of 312 (6.4%) patient viruses analyzed. The most pronounced increases in susceptibility were strongly associated with an N88S mutation in protease. All viruses that carried the N88S mutation were hypersensitive to amprenavir. Site-directed mutagenesis studies confirmed the causal role of N88S in determining amprenavir hypersensitivity. The presence of the N88S mutation and associated amprenavir hypersensitivity may be useful in predicting an improved clinical response to amprenavir salvage therapy.